Due to recent advances in biotechnology, therapeutic proteins and other biomolecules, e.g. antibodies and antibody fragments, can now be prepared on a large scale, making such biomolecules more widely available. Unfortunately, the clinical usefulness of potential therapeutic biomolecules is often hampered by their rapid proteolytic degradation, instability upon manufacture, storage or administration, or by their immunogenicity. Due to the continued interest in administering proteins and other biomolecules for therapeutic use, various approaches to overcoming these deficiencies have been explored.
One such approach which has been widely explored is the modification of proteins and other potentially therapeutic biomolecules by covalent attachment of a water-soluble polymer such as polyethylene glycol or “PEG” (Abuchowski, A., et al, J. Biol. Chem. 252 (11), 3579 (1977); Davis, S., et al., Clin. Exp Immunol., 46, 649-652 (1981). The biological properties of PEG-modified proteins, also referred to as PEG-conjugates or pegylated proteins, have been shown, in many cases, to be considerably improved over those of their non-pegylated counterparts (Herman, et al., Macromol. Chem. Phys., 195, 203-209 (1994). Polyethylene glycol-modified proteins have been shown to possess longer circulatory times in the body due to increased resistance to proteolytic degradation, and also to possess increased thermostability (Abuchowski, A., et al., J. Biol. Chem., 252, 3582-3586 (1977). A similar increase in bioefficacy is observed with other biomolecules, e.g. antibodies and antibody fragments (Chapman, A., Adv. Drug Del. Rev. 54, 531-545 (2002)).
Typically, attachment of polyethylene glycol to a drug or other surface is accomplished using an activated PEG derivative, that is to say, a PEG having at least one activated terminus suitable for reaction with a nucleophilic center of a biomolecule (e.g., lysine, cysteine and similar residues of proteins). Most commonly employed are methods based upon the reaction of an activated PEG with protein amino groups, such as those present in the lysine side chains of proteins. Polyethylene glycol having activated end groups suitable for reaction with the amino groups of proteins include PEG-aldehydes (Harris, J. M., Herati, R. S., Polym Prepr. (Am. Chem. Soc., Div. Polym. Chem), 32(1), 154-155 (1991), mixed anhydrides, N-hydroxysuccinimide esters, carbonylimadazolides, and chlorocyanurates (Herman, S., et al., Macromol. Chem. Phys. 195, 203-209 (1994)). Although many proteins have been shown to retain activity during PEG modification, in some instances, polymer attachment through protein amino groups can be undesirable, such as when derivatization of specific lysine residues inactivates the protein (Suzuki, T., et al., Biochimica et Biophysica Acta 788, 248-255 (1984)). Therefore, it would be advantageous to have additional methods for the modification of a protein by PEG using another target amino acid for attachment, such as cysteine. Attachment to protein thiol groups on cysteine may offer an advantage in that cysteines are typically less abundant in proteins than lysines, thus reducing the likelihood of protein deactivation upon conjugation to these thiol-containing amino acids.
Polyethylene glycol derivatives having a thiol-selective reactive end group include maleimides, vinyl sulfones, iodoacetamides, thiols, and disulfides. These derivatives have all been used for coupling to the cysteine side chains of proteins (Zalipsky, S. Bioconjug. Chem. 6, 150-165 (1995); Greenwald, R. B. et al. Crit. Rev. Ther. Drug Carrier Syst. 17, 101-161 (2000); Herman, S., et al., Macromol. Chem. Phys. 195, 203-209 (1994)). However, many of these reagents have not been widely exploited due to the difficulty in their synthesis and purification. For instance, the method of Woghiren, et al. (Woghiren, C., et al., Bioconjugate Chem., 4, 314-318 (1993)) requires a series of synthetic transformation and purification steps to form a particular thiol-protected PEG reagent. First, methoxy-PEG is reacted with tosyl chloride followed by a purification of the reaction product to recover the corresponding tosyl-PEG. Tosyl-PEG is then converted to the corresponding PEG-thioacetate by reaction with a thioacetate salt, followed by another purification step. Alcoholysis with methanol is then carried out on the PEG-thioacetate, followed by column chromatography to yield the purified thiolate salt, which is then reduced with dithiothreitol to form the corresponding PEG-thiol. The resulting PEG thiol is then purified by column chromatography. A protected form of the thiol is then prepared by reaction of the PEG-thiol with 4,4′-dipyridyl disulfide, followed by purification by column chromatography. In sum, Woghiren's methodology for transforming PEG to its thiol-protected form requires five different reaction steps and an additional five separate purification steps, making this and other similar synthetic approaches undesirable and extremely time-consuming.
Another significant deficiency in many of the existing routes to monofunctional thiol specific PEG derivatives is the inability, despite multiple purification steps, to remove difunctionalized PEG which arises from the diol that is present in the monofunctional PEG raw material.
Thus, there exists a need for a method for preparing high purity, activated PEG-thiols and other thiol-selective PEG derivatives that is both straightforward and simple, i.e., requiring a minimum number of reaction and purification steps, whilst maintaining the integrity of the PEG segment (i.e., is carried out under mild reaction conditions), and which can further provide high purity thiol-selective PEG derivatives in high yields. Such a method has been developed by the Applicants, to be described in greater detail below.